Methotrexate USP, commercial methotrexate, and MTX for investigational use were chromatographed by gradient elution on DEAE cellulose column. In commercial and USP methotrexate, six different peaks (excluding MTX peak) were obtained. Identity of 2 peaks were presented for the first time, using NMR and MS techniques. Percentage purity of MTX samples were found to be of the order of 86% calculated on anhydrous basis. Investigational use MTX showed a completely different spectrum of impurities, although percentage purity of the sample was the same as USP methotrexate, i.e., 86%.